Metabolic Pathway Involved in 2-Methyl-6-Ethylaniline Degradation by Sphingobium sp. Strain MEA3-1 and Cloning of the Novel Flavin-Dependent Monooxygenase System meaBA

Abstract

d 2-Methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the chloroacetanilide herbicides acetochlor and metolachlor. Sphingobium sp. strain MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol compounds as sole carbon and energy sources for growth. We isolated the mutant strain MEA3-1Mut, which converts MEA only to 2-methyl-6-ethyl-hydroquinone (MEHQ) and 2-methyl-6-ethyl-benzoquinone (MEBQ). MEA may be oxidized by the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA), which can be hydrolytically spontaneously deaminated to MEBQ or MEHQ. The MEA microbial metabolic pathway was reconstituted based on the substrate spectra and identification of the intermediate metabolites in both the wild-type and mutant strains. Plasmidome sequencing indicated that both strains harbored 7 plasmids with sizes ranging from 6,108 bp to 287,745 bp. Among the 7 plasmids, 6 were identical, and pMEA02= in strain MEA3-1Mut lost a 37,000-bp fragment compared to pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and protein mass fingerprinting (PMF) showed that MEA3-1Mut lost the two-component flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded by a gene in the lost fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with oxygenase components of some TC-FDMs, whereas MeaB showed no sequence identity with the reductase components of those TC-FDMs. Complementation with meaBA in MEA3-1Mut and heterologous expression in Pseudomonas putida strain KT2440 resulted in the production of an active MEHQ monooxygenase. Chloroacetanilide herbicides are widely used throughout the world (1) for the control of most annual grasses and certain broadleaf weeds (2). The majority of the commonly used chloroacetanilide herbicides, such as alachlor, acetochlor, butachlor, and metolachlor, are N-alkoxyalkyl-N-chloroacetyl-substituted aniline derivatives (3). Long-term application of these herbicides has caused negative impacts on both the aquatic environment and agricultural ecosystems (4, 5). Acetochlor has been classified by the U.S. Environmental Protection Agency (EPA) (4) as a B-2 carcinogen and a probable human carcinogen, and several chloroacetanilide herbicides have been proven to cause tumors in rats (6). Thus, there is great concern about the behavior and fate of chloroacetamide herbicides and their degradation metabolites in the environment.Although chloroacetamide herbicides may be degraded through chemical and physical processes, microbial metabolism is the main mechanism responsible for herbicidal degradation in natural soils (7,8). A variety of bacterial strains that are able to degrade butachlor, alachlor, acetochlor, and metolachlor have been characterized (9, 10). In the degradation pathway, these herbicides are N-dealkylated to 2-chloro-N-(2,6-diethylphenyl) acetamide (CDEPA) (for alachlor and butachlor) or 2-chloro-N-(2-methyl-6-ethylphenyl) acetamide (CMEPA) (for acetochlor and metolachlor), which are then converted to 2,6-dieth...

The Two-Component Monooxygenase MeaXY Initiates the Downstream Pathway of Chloroacetanilide Herbicide Catabolism in Sphingomonads

“…Previous studies have proposed that the aromatic ring of MEA is initially hydroxylated at the para-position in strain DE-13 (15) and Sphingobium sp. MEA3-1 (18). To verify this prediction, MeaXY-hydroxylated DEA and MEA were purified using silica gel column chromatography and subjected to nuclear magnetic resonance (NMR) analysis.…”

“…Enrichment culture techniques have been used with varied success in attempts to enrich and isolate chloroacetanilide herbicide-degrading microorganisms. A variety of bacterial strains able to degrade butachlor, alachlor, acetochlor, and metolachlor have been reported (11)(12)(13)(14)(15)(16)(17)(18). Furthermore, some bacterial pure cultures (2) or consortia (15,16) are able to mineralize these herbicides.…”

“…Using acetochlor as an example, in the upstream pathway, initial N-dealkylation is followed by amide hydrolysis to generate MEA. In the downstream pathway, MEA was hydroxylated and hydrolytically deaminated to 2-methyl-6-ethyl-hydroquinone (MEHQ); MEHQ was further hydroxylated to 3-hydroxy-2-methyl-6-ethyl-hydroquinone (3-OH-MEHQ), which is the putative substrate of ring cleavage (2,15,(18)(19)(20). Regarding the genetic basis of this pathway, a three-component Rieske nonheme iron oxygenase system, CndABC, or a cytochrome P450 system EthBAD responsible for initial N-dealkylation was identified from S. quisquiliarum DC-2 and Rhodococcus sp.…”

“…The flavindependent monooxygenase system MeaBA able to transform MEHQ to 3-OH-MEHQ was identified in Sphingobium sp. strain MEA3-1 (18). However, the genes responsible for the hydroxylation of MEA are still unknown.…”

“…In a common degradation pathway, aniline is initially converted to catechol by aniline dioxygenases (22)(23)(24). However, MEA degradation is initiated with hydroxylation at the para-position (2,15,18), which may be catalyzed by a monooxygenase.…”